1. Field of the Invention
The present invention relates to antibodies that recognize beer spoilage lactic acid bacteria capable of growing in beer, a method for detecting the beer spoilage lactic acid bacteria using the antibodies, and a kit for identification of the beer spoilage lactic acid bacteria.
2. Background of the Invention
There is a recognized danger that once a certain kind of lactic acid bacterium has contaminated the process of beer production, haze, off-flavors or the like, caused by the bacterium, may impair the quality of the produced beer. For example, representative beer spoilage bacteria include certain species of lactic acid bacterium belonging to the Lactobacillus genus, especially those belonging to Lactobacillus brevis and Lactobacillus lindneri, as well as include certain species of lactic acid bacterium belonging to the Pediococcus genus. Thus, attempts have been made to develop methods for the rapid and high sensitivity detection of these beer spoilage lactic acid bacteria.
Specifically mentioned is a method in which after the beer to be analyzed is filtrated with a membrane filter, it is grown in a suitable medium and the grown colonies are observed. However, since beer spoilage bacteria existing in beer are presented in minute quantities and their growth in the medium is slow, there are problems with the sensitivity and the time-consuming nature of their detection using this method.
A method for the detection of beer spoilage lactic acid bacteria in beer that utilizes antigen-antibody reaction has been developed. For example, polyclonal antibodies (antisera) against lactic acid bacteria have been prepared. (Sharpe, M. E., J. Gen. Microbiol., 12, 107 (1955); Sharpe, M. E., Int. J. Syst. Bacteriol., 20, 509 (1970); Knox, M. W. et al., Infect. Immun., 24, 12 (1979); Shimohashi, H. and Mutai, M., J. Gen. Microbiol., 103, 337 (1977); and Japanese Unexamined Patent Appln. Publn. Hei 4-72570.) At present, by adjusting various conditions, it has become possible to prepare antisera of such quality that both their specificity and their sensitivity are, to a certain degree, acceptable for practical use. Further improvements however remain to be desired in the following aspects, among others: the antibody titer or the specificity of an antiserum unavoidably fluctuates with its lot; antiserum against a certain kind of lactic acid bacterium turns positive against non-beer spoilage bacteria as well; and some antisera require NaOH treatment.
For these reasons, detection methods employing monoclonal antibodies have been proposed. (Japanese Unexamined Patent Appln. Publn. Hei 6-46881 and Japanese Unexamined Patent Appln. Publn. Hei 6-105698). According to the description of Japanese Unexamined Patent Appln. Publn. Hei 6-46881, monoclonal antibodies are prepared using as antigens, Lactobacillus brevis, Lactobacillus collinoides and Lactobacillus suebicus that belong to Group 3 of the Lactobacillus genus and that are grown in media; and these monoclonal antibodies are used to detect Group 3 of the Lactobacillus genus in beer.
However, the lactic acid bacteria, which will prove to grow in beer production, are only part of those belonging to Group 3 of the Lactobacillus genus. Therefore, the method as described in Japanese Unexamined Patent Appln. Publn. Hei 6-46881 has a problem that other non-beer spoilage lactic acid bacteria may also be detected, i.e., false positive results are obtained with this method.
According to the description of Japanese Unexamined Patent Appln. Publn. Hei 6-105698, a monoclonal antibody is prepared using as an antigen, Lactobacillus plantarum that belongs to the Lactobacillus genus and that is grown in medium; and this monoclonal antibody is used to detect the Lactobacillus and Lactobacillus coryniformis in beer. However, this disclosed antibody has the same problem that it also detects non-beer spoilage lactic acid bacteria (false positives).
Consequently, there has been proposed a method to detect only beer spoilage bacteria that can grow in beer (hereinafter referred to as xe2x80x9cbeer spoilage abilityxe2x80x9d). (Japanese Unexamined Patent Appln. Publn. Hei 10-104238.) According to the method as described in Japanese Unexamined Patent Appln. Publn. Hei 10-104238, an antiserum is prepared with rabbits using as an antigen, Lactobacillus brevis that possesses no glucose assimilation ability under anaerobic conditions and that is grown in medium; and the antiserum is used to detect the Lactobacillus brevis that possesses the beer spoilage ability. The thus prepared antiserum is believed to specifically react with Lactobacillus brevis and Pediococcus damnosus, both of which cause the beer spoilage ability.
Nevertheless, to remove from the antiserum nonspecific antibodies that react with Lactobacillus brevis (i.e., nonspecific antibodies that react with non-beer spoilage lactic acid bacteria), the alkali-treated cells of non-beer spoilage Lactobacillus brevis must be used to effect removal of the nonspecific antibodies by adsorption, according to the method as described in Japanese Unexamined Patent Appln. Publn. Hei 10-104238. In addition, a problem exists in that depending on the Lactobacillus brevis to be used as the antigen, the resulting antiserum is prone to false recognition. Further, now that antiserum is used, its antibody titer and specificity unavoidably fluctuates from lot to lot.
As a representative beer spoilage lactic acid bacterium mentioned is Lactobacillus lindneri; but the effectiveness of the antiserum against such bacterium is not noted in the publication of Japanese Unexamined Patent Appln. Hei 10-104238.
In view of the foregoing, improved methods of detecting bacteria which are spoilage to beer are needed in the fermentation industry.
It is an object of the present invention to provide reagents capable of detecting a lactic acid bacterium which is beer spoilage to the quality of beer.
It is another object of the present invention to provide a method for the specific, rapid, convenient, and reproducible detection of a beer spoilage lactic acid bacterium which contaminates into the process of beer production and grows in the produced beer and that degrades the quality of the beer.
It is another object of the present invention to provide a method for the efficient and exhaustive detection of a beer spoilage lactic acid bacterium such as Lactobacillus brevis, Lactobacillus lindneri, or Pediococcus damnosus. 
An additional object of the present invention is to provide an identification kit for accurately and conveniently predicting the beer spoilage ability of a lactic acid bacterium that has emerged from conventional culturing with medium.
The objects of the invention, and others, may be accomplished with an antibody for the detection of a beer spoilage lactic acid bacterium that is produced by growing a lactic acid bacterium with the beer spoilage ability in beer and by immunizing a mammal with the beer.
The objects of the invention may also be accomplished with a monoclonal antibody that displays reactivity against a lactic acid bacterium showing spoilage to beer and that displays no reactivity against a non-beer spoilage lactic acid bacterium, where the antibody is produced by growing a lactic acid bacterium with the beer spoilage ability in beer and by immunizing a mammal with the beer.
The objects of the invention may be accomplished the antibody for the detection of a beer spoilage lactic acid bacterium, as well as the monoclonal antibody as described above, where the lactic acid bacterium with beer spoilage ability is a lactic acid bacterium possessing the glucose assimilation ability under anaerobic conditions.
The objects of the invention may also be accomplished with the antibody for the detection of a beer spoilage lactic acid bacterium, as well as the monoclonal antibody as described above, where the lactic acid bacterium with the beer spoilage ability is a lactic acid bacterium selected from the group consisting of beer spoilage Lactobacillus brevis, beer spoilage Lactobacillus lindneri, and beer spoilage Pediococcus damnosus. 
The objects of the invention may also be accomplished a method for the detection of a beer spoilage lactic acid bacterium with beer spoilage ability such as Lactobacillus brevis, Lactobacillus lindneri, and Pediococcus damnosus using the antibody described above.
The objects of the invention may be accomplished a hybridoma cell line, e.g., BLb2F37 (FERM BP-6744), BG3A5b4 (FERM BP-6745), or PQ3H8a9 (FERM BP-6746), that produces the monoclonal antibody specifically reacting with a beer spoilage lactic acid bacterium.
The objects of the invention may also be accomplished with a method for the detection of a beer spoilage lactic acid bacterium with the beer spoilage ability such as Lactobacillus brevis, Lactobacillus lindneri, and Pediococcus damnosus, using a combination of the monoclonal antibodies produced by the hybridomas.
The objects of the invention may also be accomplished with a kit for the identification of a beer spoilage lactic acid bacterium, having at least the following components: (1) a centrifugation tube equipped with a filter for trapping bacteria; (2) a monoclonal antibody produced by the hybridoma according to the invention; (3) a secondary antibody or an antibody-like substance against the aforementioned antibody each of which is labeled with enzyme; and (4) a substrate reacting with and coloring the labeling enzyme of the secondary antibody or of the antibody-like substance.
The objects of the invention may also be accomplished with methods of making the antibodies and hybridomas described above.